36. Cryopreservation of primate mesenchymal stem cells with antioxidants as additional CPA

Abstract

Objectives: Stem cells have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since currently used methods exhibit poor viability and high differentiation rates. A high survival rate is a function of optimal cooling rate, appropriate cryoprotective agent (CPA) and its adjusted concentration. The most widely used CPA, ME2SO, however is toxic at high concentrations and has detrimental effects on the biological functioning of a cell. Therefore, it is of great interest to develop new cryoprotective strategies to replace currently used CPAs or to reduce their concentration. Since one of the major damaging factors is the occurrence of ROS during and after thawing we have investigated the addition of ascorbic acid and α-tocopherol as potential antioxidants. Methods: Mesenchymal stem cells (MSC) from the common marmoset monkey (Callithrix jacchus) were frozen in 200 μl PCR-tubes (5 × 105 cells/ml) with optimal cooling rate (gained from a parallel study) in a μ-freezer device. Different concentrations of ascorbic acid (50, 100 and 250 mM) and α-tocopherol (100, 200 and 500 μM) were studied exclusively or in combination with ME2SO (2%, 5% and 5% v/v). Cells attached to the culture flask surface after 24 h recultivation were considered as viable, the survival rate was compared to that of cells frozen with ME2SO as positive control. Survived cells were further tested for differentiation capability. Results: The addition of 200 μM α-tocopherol improved the survival of primate MSCs after cryopreservation even with 2%, 5% ME2SO. With 100 μM α-tocopherol and 5% ME2SO survival was more than two fold higher than with 5% ME2SO alone. Ascorbic acid had no considerable effect on the survival rate of MSCs. Intracellular lipid vesicles were clearly stained by Oil Red O and proved the adipogenic differentiation capability of marmoset MSCs after cryopreservation with antioxidant addition. Conclusions: Accessory application of α-tocopherol improved survival and proliferation of MSCs from the marmoset monkey after cryopreservation. ME2SO could be reduced to moderate concentrations. These findings will be transferred on the cryopreservation of iPS-cells in further studies.